Nonclinical pharmacokinetics and biodistribution of VSV-GP using methods to decouple input drug disposition and viral replication

Joseph Ashour, Ph.D

Abstract: Viral replication places oncolytic viruses (OVs) in a unique niche in the field of drug pharmacokinetics (PK) as their self-amplification obscures exposure-response relationships. Moreover, standard bioanalytical techniques are unable to distinguish the input from replicated drug products. Here, we combine two novel approaches to characterize PK and biodistribution (BD) after systemic administration of vesicular stomatitis virus pseudotyped with lymphocytic choriomeningitis virus glycoprotein (VSV-GP) in healthy mice. First: to decouple input drug PK/BD versus replication PK/BD, we developed and fully characterized a replication-incompetent tool virus that retained all other critical attributes of the drug. We used this approach to quantify replication in blood and tissues and to determine its impact on PK and BD. Second: to discriminate the genomic and antigenomic viral RNA strands contributing to replication dynamics in tissues, we developed an in situ hybridization method using strand-specific probes and assessed their spatiotemporal distribution in tissues. This latter approach demonstrated that distribution, transcription, and replication localized to tissue-resident macrophages, indicating their role in PK and BD. Ultimately, our study results in a refined PK/BD profile for a replicating OV, new proposed PK parameters, and deeper understanding of OV PK/BD using unique approaches that could be applied to other replicating vectors.

Targeting liver with nucleic acid therapeutics for the treatment of liver-originated systemic diseases

Xiaobo Zhong, Ph.D

Abstract: Coming soon

Light Speed Development Approaches, Which Facilitated EUA Filing with Paxlovid^TM

Innovative First-in-Human Study and Dosing Regimen Selection for Accelerated Development of Nirmatrelvir

Ravi Shankar Singh, PhD

Coronavirus disease 2019 (COVID-19) is a continued leading cause of hospitalization and death. Safe, efficacious COVID-19 antivirals are needed urgently. Nirmatrelvir (PF-07321332), the first orally bioavailable, severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) M^pro inhibitor against the coronaviridae family, has demonstrated potent preclinical antiviral activity and benign safety profile. An accelerated 5-part FIH study including single-ascending dose (SAD), Multiple-ascending dose (MAD), relative bioavailability, food effect, Japanese PK, metabolism and excretion and supratherapeutic exposure cohorts was conducted to assess safety and pharmacokinetics of nirmatrelvir. Two interleaving SAD cohorts were evaluated in a three-period crossover. MAD with nirmatrelvir/ritonavir twice daily (b.i.d.) dosing was evaluated over 10 days parallel cohorts. Safety was assessed, including in a supratherapeutic exposure cohort. Dose and dosing regimen for clinical efficacy evaluation in phase II/III clinical trials were supported by integrating modeling and simulations of SAD/MAD data with nonclinical data and a quantitative systems pharmacology model (QSP). In SAD, MAD, and supratherapeutic exposure cohorts, nirmatrelvir/ritonavir was safe and well-tolerated. Nirmatrelvir exposure and half-life were considerably increased by ritonavir, enabling selection of nirmatrelvir/ritonavir dose and regimen for phase II/III trials (300/100 mg b.i.d.), to achieve concentrations continuously above those required for 90% inhibition of viral replication in vitro. The QSP model suggested that a 5-day regimen would significantly decrease viral load in SARS-CoV-2-infected patients which may prevent development of severe disease, hospitalization, and death. In conclusion, an innovative and seamless trial design expedited establishment of phase I safety and pharmacokinetics of nirmatrelvir/ritonavir, enabling high confidence in phase II/III dose selection and accelerated pivotal trials’ initiation. The presentation will include the details of the planning and conduct of the study, pharmacokinetics and dosing regimen selection of nirmatrelvir

Metabolism and Disposition of Nirmatrelvir in Humans Using Fluorine NMR Instead of ¹⁴Carbon

R. Scott Obach, PhD

Typically human ADME studies are executed using radiolabeled (e.g., ¹⁴C) material, the synthesis of which is a time-consuming activity.  We were able to assess the metabolism and excretion of unlabeled nirmatrelvir (PF-07321332) within the first-in-human study via a novel application of quantitative fluorine NMR spectroscopy in place of a standard radiolabel ADME study. Six healthy subjects received a single 300-mg oral dose of nirmatrelvir (in combination with ritonavir), and excreta were collected up to 10 days. Virtually all drug-related material was recovered within 5 days, and mass balance was achieved with approximately 84.9 ± 8.9% (range=70.7–95.5%) of the administered dose recovered in urine and feces. Urine, fecal homogenate, and plasma pools were also analyzed for metabolite profiles by HPLC followed by 19F NMR quantitation of fractions. Metabolites were characterized and identified by HRMS. This successful demonstration of quantitative ¹⁹F NMR spectroscopy to establish the mass-balance, excretion, and metabolic profile of nirmatrelvir offers an advantageous means to execute human ADME studies for fluorine-containing compounds early in drug development.  The presentation will include specifics of how this was accomplished, in comparison to standard ¹⁴C ADME studies

“Discovery and Visualization of Uncharacterized Drug-Protein Adducts Using Mass Spectrometry”

By Dr. Alex Zelter,

Department of Biochemistry, University of Washington

Drugs are often metabolized to reactive intermediates that form protein adducts. Adducts can inhibit protein activity, elicit immune responses, and cause life-threatening adverse drug reactions. The masses of reactive metabolites are frequently unknown, rendering traditional mass spectrometry-based proteomics approaches incapable of adduct identification. Here, we present Magnum, an open-mass search algorithm optimized for adduct identification, and Limelight, a web-based data processing package for analysis and visualization of data from all existing algorithms. Limelight incorporates tools for sample comparisons and xenobiotic-adduct discovery. We validate our tools with three drug/protein combinations and apply our label-free workflow to identify novel xenobiotic-protein adducts in CYP3A4. Our new methods and software enable accurate identification of xenobiotic-protein adducts with no prior knowledge of adduct masses or protein targets. Magnum outperforms existing label-free tools in xenobiotic-protein adduct discovery, while Limelight fulfills a major need in the rapidly developing field of open-mass searching, which until now lacked comprehensive data visualization tools

“Circumventing the Blood-Testis Barrier: Transporter-Mediated Therapeutic Delivery”

By Dr. Nathan J. Cherrington,

Professor and 1885 Society Distinguished Scholar

Department of Pharmacology and Toxicology at the University of Arizona

“The Nonclinical Disposition and PK/PD Properties of GalNAc-conjugated siRNA Are Highly Predictable and Build Confidence in Translation to Man”

By Dr. Robin McDougall,

Director Quantitative Pharmacology

Repare Therapeutics 

“Drug Phosphorylation by Adenylate and Creatine Kinases”

By Dr. Namandjé N. Bumpus,

E.K. Marshall and Thomas H. Maren Professor and Director

Department of Pharmacology and Molecular Sciences

Johns Hopkins University School of Medicine

To view recording, use passcode: &.xJ3gK1

“Oral Inhibitors of the SARS-CoV-2 Main Protease for the Treatment of COVID-19”

By Dr. Dafydd Owen, Director of Medicinal Chemistry at Pfizer

“Plasma protein-mediated uptake of drugs and its impact in clearance prediction”

By Dr. David Hallifax from The University of Manchester

“How DMPK Embraces its Role in Supporting Gene Therapy”

Gene therapy technology holds an enormous promise for patients in treating a variety of diseases. The rising numbers of clinical trials undeniably speak to this ever-expanding field.  Since the approval of Luxturna and Zolgensma, recombinant adeno-associated virus (rAAV)-as delivery vehicle further confirms its utility and continues to be the leading platform for gene therapy. While the multi-disciplinary functions within each company become more knowledgeable about rAAV-based gene delivery, the roles and responsibilities for each function also becomes more defined. DMPK plays a substantial role in advancing therapeutic gene therapy modality from preclinical research to clinical development, in particular  characterization of drug product biodistribution, immunogenicity assessment, and multiple platform bioanalytical assays and biomarker analysis, as well as first in human dose projection. The overview of each role, strategic approach to existing challenges and potential mitigation plans will be discussed.

By Dr. Nancy Chen from Takeda

“Drug metabolism by human gut microbes.”

Oral medicinal drugs can exhibit incomplete absorption in the upper gastrointestinal tract or reach the gut after enterohepatic circulation. In these circumstances, drugs encounter enormous densities of commensal microbes. These microbes collectively encode 150-fold more genes than the human genome, including a rich repository of enzymes with the potential to metabolize drugs. However, the contribution of the microbiome to drug and drug metabolite exposure in the GI tract and in circulation is largely unexplored.

I will describe examples that suggest that gut microbial activity can be responsible for a significant portion of systemic exposure to a toxic drug metabolite, even if the drug exhibits high bioavailability, if the same metabolite is readily produced by hepatic extracts in vitro, and if drug metabolite levels are low in feces. I will also introduce our efforts to explore the spectrum of microbiome-encoded drug metabolizing activities and to identify microbial genes that predict the capacity of an individual’s gut microbiome to metabolize a drug.

By Dr. Andrew Goodman from Yale University School of Medicine

Andrew L. Goodman, PhD, is the C. N. H. Long Professor of Microbial Pathogenesis at Yale University School of Medicine and Director of the Yale Microbial Sciences Institute. Goodman received his undergraduate degree in Ecology and Evolutionary Biology from Princeton University, his PhD in Microbiology and Molecular Genetics from Harvard University, and completed postdoctoral training at Washington University. His lab uses microbial genetics, gnotobiotics, and mass spectrometry to understand how the gut microbiome contributes to drug metabolism. The Goodman lab works to identify and characterize microbiome-encoded drug metabolizing enzymes, and to define how these microbial activities contribute to drug and drug metabolite exposure in the gut and in circulation. The lab’s contributions have been recognized by the NIH Director New Innovator Award, the Pew Foundation Research Scholars Program, the Dupont Young Professors Award, the Burroughs Wellcome Investigator in Infectious Disease Award, the Howard Hughes Medical Institute Faculty Scholars Program, and the Presidential Early Career Award in Science and Engineering.

“The Drug Metabolism Scientist’s Contribution to the Development of Gene Therapies: It’s not what you do, it’s the way that you do it.”

By Dr. Mark Milton from Novartis

Please check back in a couple of weeks for more information about this event.